CHARACTERIZATION OF MULTIDRUG-RESISTANCE P-GLYCOPROTEIN TRANSPORT FUNCTION WITH AN ORGANOTECHNETIUM CATION

Multidrug resistance (MDR) in mammalian cells and tumors is associated with overexpression of an similar to 170 kDa integral membrane efflux transporter, the MDR1 P-glycoprotein. Hexakis(2-methoxyisobutyl isoni trile)technetium(I) (Tc-SESTAMIBI), a gamma-emitting lipophilic cation ic metallopharmaceutical, has recently been shown to be a P-glycoprote in transport substrate. Exploiting the negligible lipid membrane adsor ption properties of this organometallic substrate, we studied the tran sport kinetics, pharmacology, drug binding, and modulation of P-glycop rotein in cell preparations derived from a variety of species and sele ction strategies, including SW-1573, V79, Alex, and CHO drug-sensitive cells and in 77A, LZ-8, and Alex/A.5 MDR cells. Rapid cell accumulati on (t(1/2) approximate to 6 min) of the agent to a steady state was ob served which was inversely proportional to immunodetectable levels of P-glycoprotein. Many MDR cytotoxic agents inhibited P-glycoprotein-med iated Tc-SESTAMIBI efflux, thereby enhancing organometallic cation acc umulation. Median effective concentrations (EC(50); mu M) were as foll ows: vinblastine, 13; daunomycin, 55; idarubicin, 65; actinomycin D, 2 35; colchicine, minimal inhibition; adriamycin, no effect. P-glycoprot ein modulators generally demonstrated significantly greater potency (E C(50); mu M): SDZ PSC 833, 0.08; cyclosporin A, 1.3; verapamil, 4.1; q uinidine, 6.4; prazosin, >300. Modulator-induced enhancement up to 100 -fold was observed with Hill coefficients approximate to 1, consistent with simple Michaelis-Menten kinetics. Vanadate was an efficacious tr ansport inhibitor, while agents usually not included in the MDR phenot ype were without effect. Scatchard analysis showed quinidine to be a n oncompetitive inhibitor of P-glycoprotein-mediated Tc-SESTAMIBI transp ort, indicating allosteric effector sites on P-glycoprotein. The lipid bilayer adsorbing agents tetraphenyl berate and phloretin induced lar ge increases in final Tc-SESTAMIBI accumulation, showing maximal accum ulations 2-fold greater than classic MDR modulators and Hill coefficie nts much greater than 2. In V79 and 77A cells, modulators of PKC activ ity altered Tc-SESTAMLBI accumulation, while there was no indication o f modulation of P-glycoproteinmediated Tc-SESTAMIBI transport by hypot onic buffer, extracellular ATP, Cl-, or K+ (membrane potential). While recognized and avidly transported by the P-glycoprotein at buffer con centrations as low as 7 pM, Tc-SESTAMTBI at up to 100 mu M only minima lly modulated the cytotoxic action of colchicine, doxorubicin, or vinb lastine in MDR cells. In conclusion, transport analysis with Tc-SESTAM IBI is a sensitive assay for detecting functional expression of low le vels of P-glycoprotein and for the quantitative characterization of tr ansporter modulation and regulation. The biochemical data favor a high K-m, high capacity allosterically modulated translocation mechanism f or P-glycoprotein-mediated transport of this organometallic cation. In addition, the known physicochemical properties of Tc-SESTAMIBI combin ed with effects of the membrane adsorbing agents indicate that lipid-p rotein interactions are critical for transport of this metallopharmace utical and would suggest that this drug gains access to protein transp ort domains from the lipid phase with rapid time constants.